P1263: A SENSITIVE NGS ASSAY TO DETECT MEASURABLE RESIDUAL DISEASE (MRD) IN B-CELL LYMPHOPROLIFERATIVE DISEASES
Authors: L. Hebert, P. Shah, O. Diaz, A. A. Street, J. Li, E. Rudenko, A. M. Zlotnicki, C. Morrison, R. Bomben, Y. Huang, A. Medina Herrera, A. Wong, S. Gleen
Journal: HemaSphere (2022)

Background: MRD assessment in B-cell lymphoproliferative diseases has proven utility in assessing response to therapy, refining treatment and predicting clinical outcome in patients previously treated with a combination of treatments (Garcia-Marco et al. Haematologica 2019; Pui et al. Leukemia 2017). Next-generation sequencing (NGS) based approaches for MRD detection have provided high sensitivity and specificity to identify and track minimal residual quantities of clonal immunoglobulin heavy chain (IGH) gene rearrangements (Rawstron, Leukemia, 2016). Here, we report a sensitive NGS assay to detect MRD in B-cell malignancies such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and mantle cell lymphoma (MCL). Aims: To develop B-cell MRD Assay, a CAP/CLIA-accredited NGS-based MRD Laboratory Developed Test (LDT) to detect malignant B-cells at sensitivity up to 1.0 x 10-6. Methods: The workflow of the B-cell MRD Assay consists of two parts, sample preparation and sequencing. Within the sample preparation, DNA from 1-2 mL of bone marrow (BM) samples were isolated and concentrated to allow up to 20 µg DNA as the input for the downstream PCR. Prior to sequencing, the library was prepared during PCR using proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or Framework 3 (FR3). Followed by amplicon purification, equimolar pooling and sequencing on Illumina’s NextSeqDx® platform. The sequencing data FASTQ output file was analyzed using Invivoscribe B-Cell MRD Software-NextSeqDx®. Contrived DNA panels were prepared by diluting DNA from either cell lines or clinical BM samples (ALL, CLL, MM and MCL) into DNA from healthy BM samples. CLSI guidelines were used to design analytical studies and assess the limit of detection (LoD), limit of quantitation (LoQ), limit of blank (LoB), linearity, precision and accuracy of the B-cell MRD Assay and to determine the analytical performances of the Assay. Results: Using FR1 targeted PCR MMx, the B-Cell MRD Assay exhibited a LoB at 0, LoD at 2.3x10-6, LoQ at 2.4x10-6, good linearity with R2>0.88 across 4.5 logs and precision with %CVs (coefficient variation) ranging from 27.6% to 63.5% at the LoD. Using FR3 targeted PCR MMx, the B-Cell MRD Assay exhibited a LoB at 0, LoD at 1.8x10-6, LoQ at 1.7x10-5, good linearity with R2>0.78 across 4.5 logs and precision with %CVs ranging from 46.6% to 89.4% at the LoD. In addition, the assay demonstrated 100% agreement in an accuracy study that included 20 clinical specimens and 10 healthy donors. Summary/Conclusion: The turn-around-time (TAT) for B-Cell MRD Assay is about 5 days starting from specimens to MRD result. The assay has demonstrated good linearity, specificity and sensitivity to meet clinical threshold requirement of 10-5 (FR1 LoD is 2.3x10-6, FR3 LoD is 1.8x10-6) and showed excellent concordance in detection of patient specific clonal sequences at MRD levels. These results demonstrate that the B-Cell MRD Assay is a highly sensitive tool for detecting MRD in B-cell malignancies.